Myristoyl derivatives of 9-amino-doxycycline for targeting cancer stem cells and preventing metastasis

Abstract

9-amino-doxycycline derivatives that target cancer stem cells and inhibit cancer metastasis are disclosed. These compounds selectively target CSCs, potently inhibit tumor cell metastasis in vivo with little or no toxicity, and minimize the risk of inducing antibiotic resistance. In one embodiment, a 14-carbon fatty acid moiety is covalently attached to the free amino group of 9-amino-doxycycline. The resulting "Doxy-Myr" conjugate is more than five-fold more potent than doxycycline at inhibiting the anchorage-independent growth of MCF7 CSCs. Doxy-Myr did not affect the viability of the entire MCF7 cancer cell population or normal fibroblasts grown as 2D monolayers, demonstrating remarkable selectivity for CSCs. Doxy-Myr showed no antibiotic activity against Escherichia coli and Staphylococcus aureus. Conjugates with longer (16 carbons; palmitic acid) or shorter (12 carbons; lauric acid) fatty acid chain lengths also had similar activity.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 024,216, filed May 13, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the inhibition of mitochondrial function and eradication of cancer, particularly the inhibition of cancer stem cells (CSCs) and the prevention of metastasis or the reduction of the likelihood of metastasis, using derivatives of 9-amino-doxycycline. [Background technology]

[0003] Researchers are striving to develop new anticancer treatments. Traditional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) attempt to selectively detect and eradicate rapidly growing cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies use immunotherapy (e.g., monoclonal antibodies) that selectively bind mutated tumor antigens to rapidly growing cancer cells. Unfortunately, tumor recurrence often follows these therapies, either at the same site or at one or more different sites, indicating that not all cancer cells have been eradicated. Recurrence may be due to insufficient chemotherapy doses and / or the emergence of cancer clones resistant to therapy. Therefore, novel cancer treatment strategies are needed.

[0004] Advances in mutational analysis have enabled detailed studies of genetic mutations that occur during cancer progression. Despite knowledge of the genomic landscape, modern oncology has struggled to identify primary driver mutations across cancer subtypes. This harsh reality likely stems from the unique nature of each patient's tumor, with a single tumor potentially containing multiple distinct clonal cells. Therefore, new approaches that focus on commonalities across different cancer types are needed. Targeting metabolic differences between tumor and normal cells holds promise as a novel cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed elevated expression of over 95 mRNA transcripts associated with mitochondrial biogenesis and / or mitochondrial translation. Furthermore, over 35 of the 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs). Similarly, proteomic analysis of human breast cancer stem cells revealed significant overexpression of multiple mitoribosomal proteins and other proteins associated with mitochondrial biogenesis.

[0005] Mitochondria are highly dynamic organelles that constantly divide, elongate, and connect with each other to form tubular networks or fragmented granules to meet cellular requirements and adapt to the cellular microenvironment. The balance between mitochondrial fusion and fission determines mitochondrial morphology, abundance, function, and spatial distribution, thereby affecting many mitochondria-dependent biological processes, such as ATP production, mitophagy, apoptosis, and calcium homeostasis. Mitochondrial dynamics can be regulated by mitochondrial metabolism, respiration, and oxidative stress. Therefore, it is not surprising that an imbalance between fission and fusion activity adversely affects multiple pathological conditions, including cancer. Cancer cells often exhibit fragmented mitochondria, and enhanced or decreased fission is often associated with cancer, but a comprehensive mechanistic understanding of how mitochondrial dynamics influences tumorigenesis remains necessary.

[0006] Intact and enhanced metabolic function is necessary to support the elevated bioenergetic and biosynthetic demands of cancer cells, especially as they pursue tumor growth and metastatic dissemination. Not surprisingly, mitochondrial-dependent metabolic pathways provide a biochemical platform for cancer cells by extracting energy from multiple fuel sources.

[0007] Cancer stem cells are a relatively small subpopulation of tumor cells that share distinctive properties with normal adult and embryonic stem cells. Therefore, CSCs are considered the "primary biological cause" of tumor regeneration and spread throughout the body, leading to the clinical hallmarks of tumor recurrence and distant metastasis, ultimately causing treatment failure and early death in cancer patients undergoing chemotherapy and radiotherapy. Evidence suggests that CSCs also function in tumor initiation, as isolated CSCs have been shown to behave as tumor-initiating cells (TICs) in preclinical animal models. Because approximately 90% of all cancer patients worldwide die prematurely from metastatic disease, there is a strong urgency and unmet clinical need for the development of novel therapies to effectively target and eradicate CSCs. Most conventional therapies do not target CSCs and often increase their frequency in the primary tumor and at distant sites.

[0008] Recently, energy metabolism and mitochondrial function have been linked to specific dynamics involved in the maintenance and proliferation of CSCs, a distinctive cell subpopulation within tumor masses involved in tumor initiation, metastatic spread, and resistance to anticancer therapy. For example, CSCs exhibit a specific and distinctive increase in mitochondrial mass, as well as enhanced mitochondrial biogenesis and stronger activation of mitochondrial protein translation. These behaviors suggest a strict dependence on mitochondrial function. Consistent with these observations, elevated mitochondrial metabolic function and OXPHOS have been detected in CSCs of multiple tumor types.

[0009] One emerging strategy for eliminating CSCs exploits cellular metabolism. CSCs are among the most energy-rich cancer cells. This approach uses metabolic inhibitors to induce ATP depletion, starving CSCs. To date, we have identified numerous FDA-approved drugs with anti-CSC properties and off-target mitochondrial side effects that induce ATP depletion, including doxycycline, an antibiotic that functions as a mitochondrial protein translation inhibitor. Doxycycline, a long-acting tetracycline analog, is currently used to treat various forms of infection, particularly acne, rosacea, and malaria prophylaxis. In a recent phase II clinical trial, preoperative oral doxycycline (200 mg / day for 14 days) reduced CSC burden in early-stage breast cancer patients by 17.65%–66.67%, with a positive response rate approaching 90%.

[0010] However, the use of antimitochondrial agents alone in cancer therapy has certain limitations, as tumor masses can overcome mitochondrial dysfunction by adopting adaptive mechanisms. These adaptive mechanisms include, for example, the ability of CSCs to shift from oxidative metabolism to alternative energy pathways in a multidirectional process of metabolic plasticity driven by both intrinsic and extrinsic factors within tumor cells and the surrounding niche. Manipulating such metabolic flexibility, particularly in CSCs, can be advantageous from a therapeutic perspective. Therefore, what is needed are therapeutic approaches that either prevent these metabolic shifts or otherwise exploit them to inhibit cancer cell proliferation.

[0011] Furthermore, various anticancer drugs have been described that also possess some antibiotic activity. For example, various repurposed antibiotics have been identified that have CSC inhibitory properties. While such compounds have the potential to be used as part of cancer treatment, they raise concerns about the rise of antibiotic resistance. Therefore, what is needed are treatment options that lack antibiotic activity and are therefore less likely to contribute to antibiotic resistance. Summary of the Invention [Problem to be solved by the invention]

[0012] The purpose of this disclosure is to describe pharmaceutical compounds that are designed to specifically target and eradicate cancer cells, and more particularly, cancer cells.

[0013] Another object of the present disclosure is to describe pharmaceutical compounds that are designed to specifically target CSCs involved in metastasis and tumor recurrence, and that do not have antibiotic activity.

[0014] Another object of the present disclosure is to identify novel anti-cancer therapeutic approaches and treatments, more particularly for preventing metastasis and tumor recurrence and / or reducing the likelihood of metastasis and tumor recurrence. [Means for solving the problem]

[0015] The present approach relates to a family of 9-amino-doxycycline derivatives that specifically target cancer stem cells and inhibit cancer metastasis and recurrence. The compounds disclosed herein potently inhibit tumor cell metastasis in vivo with little or no toxicity. These compounds are suitable for use in therapy to selectively target CSCs and prevent or reduce the likelihood of metastasis and recurrence, while effectively minimizing the risk of inducing antibiotic resistance. In one embodiment, a 14-carbon fatty acid moiety is covalently attached to the free amino group of 9-amino-doxycycline. The resulting "Doxy-Myr" conjugate is more than five-fold more potent than doxycycline in terms of IC50 inhibition of anchorage-independent growth of MCF7 breast CSCs. Doxy-Myr did not affect the viability of the entire MCF7 cancer cell population or normal fibroblasts grown as a 2D monolayer, demonstrating remarkable selectivity for CSCs. Doxy-Myr showed no antibiotic activity against Escherichia coli and Staphylococcus aureus when used with both Gram-negative and Gram-positive bacterial strains. Therefore, compounds of the present approach are unlikely to induce antibacterial resistance to doxycycline, a frontline antibiotic. Conjugates with longer (16 carbons; palmitic acid) or shorter (12 carbons; lauric acid) fatty acid chain lengths had similar activity but were less potent than Doxy-Myr in targeting CSCs.

[0016] The present approach relates to the chemical synthesis and biological activity of novel 9-amino-doxycycline derivatives modified with a fatty acid moiety at the 9-position to improve efficacy in targeting CSCs and preventing metastasis and reducing metastatic potential. An embodiment of the present approach is a compound having the general formula shown below or a pharmaceutically acceptable salt thereof (e.g., monohydrate, hydrochloride hydrate, etc.), where R is C4-C 18 Alkyl, preferably straight chain alkyl, preferably saturated alkyl.

[0017] [ka]

[0018] The inventive approach involves the synthesis of compounds of the general formula:

[0019] [ka]

[0020] or a pharmaceutically acceptable salt thereof, wherein R is a straight chain saturated alkyl having 4 to 18 carbons. In some embodiments, R is a straight chain saturated alkyl having 11 to 16 carbons. For example, in some embodiments, the compound has the formula:

[0021] [ka] may have

[0022] In some embodiments, the compound has the formula:

[0023] [ka] may have

[0024] In some embodiments, the compound has the formula:

[0025] [ka] may have

[0026] In embodiments in which the compound is a pharmaceutically acceptable salt, the salt may be, for example, one of a monohydrate and a hydrochloride hydrate.

[0027] The inventive approach involves the synthesis of compounds of the general formula:

[0028] [ka]

[0029] The compound may take the form of a pharmaceutical composition comprising a compound having a pharmaceutically acceptable salt thereof, wherein R is a linear saturated alkyl having 4 to 18 carbon atoms, and a pharmaceutically acceptable carrier. In some embodiments, R may be a linear saturated alkyl having 11 to 16 carbon atoms. For example, R may be 11, 13, or 15. The pharmaceutically acceptable carrier may include one or more of sugars, starches, celluloses, excipients, oils, glycols, polyols, esters, agar, and buffers. It should be understood that one skilled in the art can determine an appropriate pharmaceutically acceptable carrier without undue burden using routine means available in the art.

[0030] In some embodiments, the pharmaceutical composition may be for use in one of preventing metastasis, reducing inflammation, reducing fibrosis, and reducing viral replication.

[0031] The approach of the present invention may take the form of a method for preventing metastasis in a patient, said method comprising the step of administering to said patient a pharmaceutically effective amount of a compound described herein.

[0032] The inventive approach may take the form of a method for reducing inflammation in a patient, said method comprising administering to said patient a pharmaceutically effective amount of a compound described herein.

[0033] The approach of the present invention may take the form of a method for the reduction of fibrosis in a patient, said method comprising the step of administering to said patient a pharmaceutically effective amount of a compound described herein.

[0034] The approach of the present invention may take the form of a method for reducing viral replication in a patient, said method comprising the step of administering to said patient a pharmaceutically effective amount of a compound described herein.

[0035] These and other embodiments will be apparent to those skilled in the art in view of this description, the appended claims, and the applications incorporated by reference into this application. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows the chemical structures of exemplary 9-amino-doxycycline derivatives, (A) Doxy-Myr and (B) Doxy-TPP. [Figure 2] FIG. 2 shows the results of a 3-D tumor formation assay for one embodiment of the inventive approach. [Figure 3] Figure 3 shows comparative images of compounds that fluoresce inside cells. [Figure 4A] 4A and 4B show the cell viability results of treatment of MCF7 cells and normal human fibroblasts (hTERT-BJ1) with doxycycline ("Doxy") or Doxy-Myr. [Figure 4B] 4A and 4B show the cell viability results of treatment of MCF7 cells and normal human fibroblasts (hTERT-BJ1) with doxycycline ("Doxy") or Doxy-Myr. [Figure 5A] 5A-5D show the results of treatment with doxycycline or Doxy-Myr on 2D monolayer growth of MCF7. [Figure 5B] 5A-5D show the results of treatment with doxycycline or Doxy-Myr on 2D monolayer growth of MCF7. [Figure 5C] 5A-5D show the results of treatment with doxycycline or Doxy-Myr on 2D monolayer growth of MCF7. [Figure 5D] 5A-5D show the results of treatment with doxycycline or Doxy-Myr on 2D monolayer growth of MCF7. [Figures 6A-6C] 6A-6C show the results of the effect of treatment with doxycycline or Doxy-Myr on cell cycle progression in the form of representative FACS cell cycle profiles. [Figure 7] FIG. 7 shows the results for doxycycline (solid line), Doxy-Pal (dotted line), Doxy-Laur (dashed dotted line), and Doxy-Myr (solid line). [Figure 8A] 8A-8D show the antibiotic effects of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal at various concentrations against Escherichia coli and Staphylococcus aureus, respectively. [Figure 8B] 8A-8D show the antibiotic effects of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal at various concentrations against Escherichia coli and Staphylococcus aureus, respectively. [Figure 8C] 8A-8D show the antibiotic effects of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal at various concentrations against Escherichia coli and Staphylococcus aureus, respectively. [Figure 8D] 8A-8D show the antibiotic effects of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal at various concentrations against Escherichia coli and Staphylococcus aureus, respectively. [Figure 9] FIG. 9 shows the results regarding metastasis by CAM assay. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following description describes embodiments of the inventive approach in sufficient detail to enable the inventive approach to be practiced. While the inventive approach will be described with reference to these specific embodiments, it will be understood that the inventive approach may be embodied in different forms, and this description should not be construed as limiting any appended claims to the specific embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive approach to those skilled in the art.

[0038] This description uses various terms that will be understood by those of ordinary skill in the art. The following explanations are provided to avoid any misunderstanding. The terms "treat," "treated," "treating," and "treatment" include the reduction or amelioration of at least one symptom associated with or caused by the condition, disorder, or disease being treated, particularly cancer. In certain embodiments, treatment involves reducing and / or ameliorating at least one symptom associated with or caused by the cancer being treated with a compound of the present invention. In some embodiments, treatment involves causing the death of certain categories of cells, such as CSCs, that may be involved in metastasis or recurrence of a particular cancer in the host's body. This can be achieved by preventing further spread of cancer cells and / or inhibiting CSC function, for example, by depriving these cells of energy-producing mechanisms. For example, treatment can include the reduction of one or more symptoms of cancer, or the complete eradication of cancer. As another example, the approach of the present invention can be used to inhibit mitochondrial metabolism in cancer, eradicate CSCs in cancer (killing them at a rate faster than their rate of proliferation), eradicate TICs in cancer, eradicate circulating tumor cells in cancer, inhibit cancer growth, target and inhibit CSCs, target and inhibit circulating tumor cells, prevent (i.e., reduce the likelihood of) metastasis, prevent recurrence, sensitize cancer to chemotherapeutic agents, sensitize cancer to radiation therapy, and sensitize cancer to phototherapy.

[0039] The terms "cancer stem cells" and "CSCs" refer to a subpopulation of cancer cells within a tumor that have the capacity for self-renewal, differentiation, and tumorigenesis when transplanted into an animal host. Compared to "bulk" cancer cells, CSCs have a larger mitochondrial mass, enhanced mitochondrial biogenesis, and more active mitochondrial protein translation. As used herein, "circulating tumor cells" are cancer cells that shed from the primary tumor into the vasculature or lymphatics and are transported throughout the body in the blood circulation. The CellSearch Circulating Tumor Cell Test can be used to detect circulating tumor cells.

[0040] As used herein, the phrase "pharmaceutically effective amount" refers to the amount that needs to be administered to a host, or to a cell, tissue, or organ of a host, to achieve a therapeutic result, such as regulating, modulating, or inhibiting protein kinase activity, e.g., inhibiting the activity of a protein kinase, or treating cancer. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of the compounds of the invention contained in the pharmaceutical composition at levels below those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0041] As used herein, the phrase "active compound" refers to a 9-amino-doxycycline derivative compound described herein, which may include pharmaceutically acceptable salts or isotopic analogs thereof. It should be understood that one or more active compounds can be administered to a subject by any suitable approach, as known to those of ordinary skill in the art. It should also be understood that the amount of active compound and the timing of its administration may depend on the individual subject being treated (e.g., age and weight, among other factors), the method of administration, the pharmacokinetic properties of the particular active compound(s), and the judgment of the prescribing physician. Thus, due to subject-to-subject variability, any dosages described herein are intended as initial guidelines, and a physician can titrate the compound dose to achieve the treatment he or she deems appropriate for the subject. In considering the degree of treatment desired, a physician can balance various factors, such as the subject's age and weight, the presence of pre-existing conditions, and the presence of other conditions. As described in more detail below, pharmaceutical formulations can be prepared for any desired route of administration, including but not limited to oral, intravenous, or aerosol administration.

[0042] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) (11) glycols such as propylene glycol; (12) polyols such as glycerin, sorbitol, mannitol, polyethylene glycol, and the like; (13) esters such as ethyl oleate and ethyl laurate; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffered saline; and (21) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0043] As used herein, the term "derivative" refers to a chemical moiety derived from or synthesized from a referenced chemical moiety. For example, a compound according to the inventive approach may be referred to as a 9-amino-doxycycline derivative, having a fatty acid moiety conjugated at the 9-position. As used herein, a "conjugate" refers to a compound formed by the joining of two or more chemical compounds. For example, the conjugation of doxycycline with a fatty acid results in a compound having a doxycycline moiety and a moiety derived from the fatty acid. As used herein, a fatty acid is a carboxylic acid having an aliphatic chain, either saturated or unsaturated. Examples of fatty acids include short-chain (i.e., having five or fewer carbon atoms in its chemical structure) fatty acids, medium-chain (i.e., having 6-12 carbon atoms in its chemical structure) fatty acids, and other long-chain (i.e., having 13-21 carbon atoms in its chemical structure) fatty acids. An example of a saturated fatty acid is lauric acid (CH3(CH2)). 10 COOH), palmitic acid (CH3(CH2) 14 COOH), stearic acid (CH3(CH2) 16 COOH), and myristic acid (CH3(CH2) 12 Oleic acid (CH3(CH2)7CH=CH(CH2)7COOH) is an example of a naturally occurring unsaturated fatty acid. It is understood that the compounds of the present approach include 9-amino-doxycycline conjugated at the 9-position to a straight-chain saturated fatty acid, preferably having 5 to 19 carbon atoms, more preferably 10 to 18 carbon atoms, and even more preferably 12 to 16 carbon atoms. In a preferred embodiment, the straight-chain saturated fatty acid is myristic acid, which has 14 carbon atoms.

[0044] The present approach relates to the chemical synthesis and biological activity of novel 9-amino-doxycycline derivatives modified with a fatty acid moiety at the 9-position to improve their efficacy in targeting CSCs and preventing metastasis and reducing metastatic potential. An embodiment of the present approach is a compound having the general formula [1] or a pharmaceutically acceptable salt thereof (e.g., monohydrate, hydrochloride hydrate, etc.), wherein R is C4-C 18 Alkyl, preferably straight chain alkyl, preferably saturated alkyl.

[0045] [ka]

[0046] In certain preferred embodiments, the compound is a 9-amino-doxycycline derivative in which a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-doxycycline at position 9. The resulting compound is referred to herein for brevity as "Doxy-Myr" and is shown below as compound [1A]. Other exemplary preferred embodiments include a 9-amino-doxycycline derivative in which a lauric acid (12 carbon) moiety is attached to the amino acid group at position 9, and a 9-amino-doxycycline derivative in which a palmitic acid (16 carbon) moiety is attached to the amino acid group at position 9.

[0047] [ka]

[0048] Disclosed herein are various data demonstrating the efficacy of Doxy-Myr using a 3D tumor-like mass assay and its inhibitory effect on the anchorage-independent growth of breast CSCs. Overall, Doxy-Myr is more than five times more potent than doxycycline. Furthermore, Doxy-Myr exhibited superior intracellular retention and was specifically localized within the perinuclear membrane compartment. In contrast, when MCF7 breast cancer cells or normal fibroblasts were grown as 2D monolayers, Doxy-Myr had no effect on cell viability or proliferation, highlighting the unique selectivity of the compound for targeting the 3D growth of CSCs. Using MDA-MB-231 cells in a CAM assay, Doxy-Myr was found to potently inhibit tumor cell metastasis in vivo with little or no chick embryo toxicity. Similar effects were obtained with other 9-amino-doxycycline conjugates with longer alkyl chains (e.g., 16 carbons, palmitic acid) and shorter alkyl chains (e.g., 12 carbons, lauric acid). Although effective, the data indicated that Doxy-Myr, a conjugate with a 14-carbon alkyl chain, was the most potent for targeting CSCs.

[0049] The data presented herein demonstrate that a lipophilic amide substituent at position 9 of the tetracycline skeleton results in a loss of its antibacterial activity. Previously published structure-activity studies have shown that chemical modifications of the tetracycline skeleton at position 9, as exemplified by the antibiotic tigecycline, can be tolerated, leading to diverse antibacterial activities. The lipophilicity of tetracyclines appears to play an important role in the biological efficacy of this family of drugs.

[0050] The improved biological properties of 9-amino-doxycycline derivatives with respect to targeting CSCs and the associated antibacterial activity make these novel compounds highly useful in the treatment of cancer without raising concerns about antibiotic resistance or adverse effects on the human microbiome.

[0051] One previous study successfully utilized the parent compound, doxycycline, to prevent bone metastasis in a mouse model using MDA-MD-231 cells. Duivenvoorden WC, Popovic SV, Lhotak S, Seidlitz E, Hirte HW, Tozer RG, Singh G. Doxycycline reduces tumor burden in a bone metastasis model of human breast cancer. Cancer Res. 2002 Mar 15;62(6):1588-91. However, this study did not investigate the effect of doxycycline on tumor growth and focused only on bone metastasis. The study attributed doxycycline's effectiveness to its tropism for bone and its ability to act as a zinc chelator, thereby acting as a protease inhibitor of lysosomal cysteine ​​proteinases, cathepsins, and MMPs.

[0052] In contrast, the present approach demonstrates that doxycycline and 9-amino-doxycycline derivatives, such as Doxy-Myr, act as inhibitors of metastasis by targeting the 3D anchorage-independent growth of CSCs. This mechanism is completely different from that of bone metastasis. Therefore, based on these functional observations, it may be more appropriate to refer to tumorspheres as metastaspheres to better reflect the close relationship between 3D anchorage-independent growth and metastasis.

[0053] Doxycycline is known to function as an inhibitor of CSC proliferation through its ability to inhibit small mitochondrial ribosomes, an off-target side effect. Doxycycline is typically used as a broad-spectrum antibiotic with bacteriostatic properties to combat numerous infectious pathogens, including Gram-negative and Gram-positive bacteria. Therefore, the present inventors sought to derive a novel chemical entity that selectively targets CSCs by optimizing doxycycline's ability to target CSCs while minimizing its antibiotic activity.

[0054] An embodiment of the inventive approach uses 9-amino-doxycycline (shown below) as a scaffold. This 9-amino-doxycycline compound, formally known as (4S,5S,6R,12aS)-9-amino-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide, is a synthetic chemical often used in the synthesis of pharmaceutical and other organic compounds. The amine group at the 9-position (of what is known in the art as the D ring) is a useful substituent in the compounds of the inventive approach, enabling conjugates with little or no antibiotic activity.

[0055] [ka]

[0056] Substitution according to the present approach can be performed at the primary amine on the D ring. In a first embodiment, we covalently attached a linear saturated 14-carbon fatty acid moiety (myristic acid) to 9-amino-doxycycline at this position. This compound is called Doxy-Myr. For comparison, we synthesized a 6-carbon spacer arm terminated in triphenylphosphonium (TPP) at the same position to 9-amino-doxycycline, which is called Doxy-TPP. Figure 1 shows the chemical structures of these 9-amino-doxycycline derivatives.

[0057] The addition of a fatty acid moiety (e.g., myristic acid) acts as a membrane targeting signal, resulting in increased retention of Doxy-Myr within membrane compartments such as the plasma membrane, endoplasmic reticulum (ER), Golgi apparatus, and / or mitochondria. In contrast, the TPP moiety was predicted to increase the membrane potential of the compound, targeting it to mitochondria in CSCs.

[0058] To determine the functional activity of the Doxy-Myr and Doxy-TPP compounds, we used a 3D tumor formation assay to assess the ability of each compound to inhibit the anchorage-independent growth of MCF7 CSCs. Figure 2 shows the results for both doxycycline and Doxy-Myr. Doxy-Myr was more than five-fold more potent than doxycycline, with an IC 50 In contrast, the IC of doxycycline was 3.46 μM. 50 was 18.1 μM. This demonstrates that Doxy-Myr is more potent in targeting the 3D anchorage-independent growth of CSCs. Because Doxy-TPP was not more potent than doxycycline itself, further assays with Doxy-TPP were not performed. Data on Doxy-TPP are not shown.

[0059] Further evaluation demonstrated that Doxy-Myr is better retained within cells than doxycycline. Because doxycycline and Doxy-Myr are fluorescent (Ex. 390-425 nm / Em. 520-560 nm), their cellular retention can be visually compared. Figure 3 shows images of the compounds fluorescing in MCF7 cell monolayers. As can be seen, Doxy-Myr is more readily detected and retained within MCF7 cell monolayers compared to both doxycycline and vehicle-only treated cells. Doxy-Myr fluorescence exhibits a perinuclear staining pattern consistent with its partitioning and retention in intracellular membrane compartments. This observation may explain the mechanism behind Doxy-Myr's increased efficacy. No nuclear staining of Doxy-Myr was observed, indicating that most of this compound was excluded from the nucleus.

[0060] Embodiments of the present approach have been demonstrated to be non-toxic to normal fibroblasts. For example, this Doxy-Myr embodiment was found to be non-toxic to 2D monolayers of MCF7 cells or normal human fibroblasts. MCF7 cells and normal human fibroblasts (hTERT-BJ1) were treated over a 3-day period to assess toxicity.

[0061] Figures 4A and 4B show the results of cell viability when MCF7 cells and normal human fibroblasts (hTERT-BJ1) were treated with doxycycline ("Doxy") or Doxy-Myr. The cells were grown as 2D monolayers and treated for a 3-day period. At the concentrations tested, Doxy-Myr does not affect the viability of MCF7 cells or normal fibroblasts when grown as 2D monolayers. As can be seen, doxycycline and Doxy-Myr had no apparent effect on cell viability in either cell line over the concentration range of 5 to 20 μM.

[0062] Potential 2D effects on cell proliferation and cell cycle were also determined using MCF7 cell monolayers. Figures 5A-5D show the results of doxycycline or Doxy-Myr treatment on MCF7 2D monolayers, as assessed using xCELLigence. These results are shown relative to the control (no treatment). Figures 5A and 5B show the results for doxycycline, and Figures 5C and 5D show the results for Doxy-Myr. As can be seen, treatment with either doxycycline or Doxy-Myr did not inhibit MCF7 cell proliferation compared to the control (no treatment).

[0063] Figures 6A-6C show the effects of doxycycline or Doxy-Myr treatment on cell cycle progression in the form of representative FACS cell cycle profiles. MCF7 cells were cultured as 2D monolayers for 72 hours in the presence of 10 μM doxycycline (Figure 6B) or Doxy-Myr (Figure 6C). A vehicle-only control was treated in parallel (Figure 6A). Compared to the parent compound, doxycycline, Doxy-Myr had no significant effect on reducing cell cycle progression in 2D monolayers of MCF7 cells.

[0064] Overall, these results indicate that Doxy-Myr did not significantly reduce cell viability, proliferation, or cell cycle progression of 2D monolayers of MCF7 cells, indicating that the effect of Doxy-Myr is specific to cell proliferation under 3D anchorage-independent growth conditions.

[0065] Embodiments of the present approach have improved CSC inhibitory effects compared to doxycycline. Data indicate that this effect depends on the length of the linear saturated alkyl chain. Two embodiments of 9-amino-doxycycline conjugated at the 9 position with lauric acid (12-carbon chain) ("Doxy-Laur") and palmitic acid (16-carbon chain) ("Doxy-Pal"), shown below as compounds [1B] and [1C], respectively, were synthesized and evaluated. Both 9-amino-doxycycline conjugates were found to be weaker than Doxy-Myr in targeting CSCs.

[0066] [ka]

[0067] A 3D tumor mass assay was used to compare the functional inhibitory activity of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal using MCF7 cells. Figure 7 shows the results for doxycycline (solid line), Doxy-Pal (dotted line), Doxy-Laur (dashed-dotted line), and Doxy-Myr (solid line). These results indicate that all three 9-amino-doxycycline conjugates had improved inhibitory activity compared to doxycycline. As can be seen, the IC of Doxy-Myr was 0.01. 50 The IC of Doxy‐Laur was 3.46 μM. 50 The IC of Doxy‐Pal was 5.8 μM. 50 is 10.4 μM, and the IC of doxycycline 50was 3.46 μM. Figure 7 demonstrates that embodiments of the present approach are effective in inhibiting CSC proliferation, with the myristoyl derivative of 9-amino-doxycycline being the most potent. The rank order of potency is: Doxy-Myr > Doxy-Laur > Doxy-Pal > Doxycycline, with no direct correlation observed between chain length and activity. Thus, conjugation to a 14-carbon myristic acid moiety appears to be the optimal chain length modification for embodiments of the present approach.

[0068] Embodiments of the present approach appear to lack antibiotic activity against common Gram-negative and Gram-positive bacteria. The lack of antibiotic activity reduces or eliminates concerns about the potential development of antibiotic resistance, which can be a concern when using frontline antibiotics such as doxycycline in anti-cancer therapy. Doxycycline is a well-established broad-spectrum antibiotic that is routinely used to therapeutically target both Gram-negative and Gram-positive bacterial infections. The antibiotic activity of fatty acid derivatives of 9-amino-doxycycline from the present approach was evaluated.

[0069] Figures 8A-8D show the antibiotic effects of doxycycline, Doxy-Myr, Doxy-Laur, and Doxy-Pal against E. coli and S. aureus, respectively, at various concentrations. As expected, doxycycline potently and effectively inhibited the growth of both Gram-negative (E. coli) and Gram-positive (S. aureus) microorganisms at most of the concentrations evaluated. However, in stark contrast, Doxy-Myr, Doxy-Laur, and Doxy-Pal showed no antibiotic activity over the same concentration range. Thus, chemical modification of 9-amino-doxycycline according to the present approach eliminated antibiotic activity while simultaneously enhancing specificity for targeting and inhibiting CSCs.

[0070] Embodiments of the inventive approach inhibit cancer cell metastasis without significant toxicity. These functional effects were evaluated experimentally in vivo. Tumor growth and metastasis were quantitatively measured using MDA-MB-231 cells and the well-established chicken egg chorioallantoic membrane (CAM) assay. MDA-MB-231 breast cancer cells were used for in vivo studies because they are estrogen-independent, inherently aggressive, form relatively large tumors, and are highly migratory, invasive, and metastatic. Thus, they represent a relatively excellent in vivo model for assessing both tumor growth and spontaneous metastasis. Doxycycline has been shown to effectively inhibit the 3D anchorage-independent growth of MDA-MB-231 cells, making it ideal for evaluating embodiments of the inventive approach.

[0071] 1×10 6 An inoculum of MDA-MB-231 cells was added to the CAM of each egg (day E9), after which the eggs were randomly divided into groups. Tumors were detectable on day E10, and these tumors were treated daily for 8 days with vehicle alone (1% DMSO in PBS), doxycycline, or Doxy-Myr. Eight days after drug administration, on day E18, all tumors were weighed, and the lower CAM was harvested to assess the number of metastatic cells, analyzed by qPCR using primers specific for human Alu sequences.

[0072] Both doxycycline and Doxy-Myr demonstrated significant effects on metastasis of MDA-MB-231 cancer cells. Figure 9 shows the results for metastasis using the CAM assay. These results are shown relative to the control (no treatment). As can be seen, doxycycline inhibited metastasis by 44% to 57.5%. In contrast, Doxy-Myr inhibited metastasis by 85% to 87% at the same concentrations tested for doxycycline. This demonstrates that Doxy-Myr is significantly more effective than doxycycline at preventing metastasis or reducing the likelihood of metastasis.

[0073] Furthermore, little or no embryotoxicity was observed for doxycycline and Doxy-Myr in the CAM assay. Doxy-Myr has efficacy as an anti-metastatic agent, selectively inhibiting tumor metastasis without significant toxicity or antibiotic activity. Table 1 below summarizes the toxicity analysis from the CAM assay.

[0074] [Table 1]

[0075] Although the data disclosed herein are primarily based on breast cancer (e.g., MCF7 and hTERT-BJ1 cell lines), the compounds of the inventive approach are also effective against other types of cancer. In previous studies, the inventors demonstrated that mitochondrial biogenesis inhibitors successfully inhibit tumorsphere formation in a wide range of cell lines from multiple tumor types. Table 2 below lists cancer cell lines that have been shown to be sensitive to mitochondrial biogenesis inhibitors. These results suggest that the inventive approach is effective against many types of cancer.

[0076] [Table 2]

[0077] The above few paragraphs demonstrate the efficacy of 9-amino-doxycycline derivatives bearing a fatty acid moiety as anticancer therapeutic agents, more specifically, for preventing or reducing the likelihood of metastasis. In addition to inhibiting and eradicating CSC metastasis, the compounds of the present approach also have efficacy as anti-inflammatory, antifibrotic, and antiviral agents. Doxycycline was originally shown to act as an inhibitor of bacterial protein synthesis. As a result, doxycycline also inhibits mammalian cell protein synthesis as an off-target side effect.

[0078] As a result of its ability to inhibit protein synthesis, doxycycline can also act as an anti-inflammatory agent by reducing the synthesis and secretion of IL-6 and other cytokines, including IL-1β and TNF-α in particular. Doxycycline can also inhibit collagen synthesis and secretion, thereby inhibiting fibrosis. Finally, doxycycline inhibits viral replication of dengue virus and other viruses, since these viruses are composed of proteins.

[0079] Although compounds based on the present approach, such as Doxy-Myr, share many of these properties with doxycycline, Doxy-Myr is a more potent inhibitor of protein synthesis. Interestingly, the perinuclear localization pattern of Doxy-Myr within cells is reminiscent of the endoplasmic reticulum (ER), a major site of protein synthesis for inflammatory cytokines, collagen isoforms, and viral spike glycoproteins. Therefore, the improved efficacy of Doxy-Myr in reducing metastasis may also be explained by its effect on protein synthesis in CSCs.

[0080] Importantly, the use of Doxy-Myr in combating viral infections by inhibiting viral replication may have broad applicability, particularly for its use in emerging viral pandemics, such as the current COVID-19 pandemic of 2020, where a vaccine is still not available or has yet to be developed.

[0081] It should be understood that some embodiments of the present approach can take the form of a pharmaceutical composition, such as a composition for preventing metastasis and / or reducing the likelihood of metastasis. The pharmaceutical composition of the present approach comprises a 9-amino-doxycycline derivative (including its salt) as the active compound in any pharmaceutically acceptable carrier. When a solution is desired, water can be the carrier of choice for water-soluble compounds or their salts. For water solubility, organic vehicles such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof can be suitable. Furthermore, methods for improving water solubility can be used without departing from the present approach. In the latter case, the organic vehicle can contain a significant amount of water. In either case, the solution can be sterilized by suitable means known in the art, for example, filtration through a 0.22 micrometer filter. After sterilization, the solution can be dispensed into suitable containers, such as pyrogen-free glass vials. This dispensing is optionally performed in an aseptic manner. A sterilized closure can then be placed on the vial, and the contents of the vial can be lyophilized, if desired. The approach of the present invention is not intended to be limited to any particular form of administration unless otherwise specified.

[0082] In addition to the active compound, the pharmaceutical formulations of the present approach can contain other additives known in the art. For example, some embodiments may include pH adjusters, such as acids (e.g., hydrochloric acid) and bases or buffers (e.g., sodium acetate, sodium borate, sodium citrate, sodium gluconate, sodium lactate, sodium phosphate). Some embodiments may include antimicrobial preservatives, such as methylparaben, propylparaben, and benzyl alcohol. Antimicrobial preservatives are often included when the formulation is packaged in vials designed for multiple doses. The pharmaceutical formulations described herein can be lyophilized using techniques known in the art.

[0083] In embodiments involving oral administration of the active compound, the pharmaceutical compositions may take the form of capsules, tablets, pills, powders, solutions, suspensions, and the like. Tablets containing various excipients, such as sodium citrate, calcium carbonate, calcium phosphate, and the like, may be used with a variety of disintegrants, such as starch (e.g., potato or tapioca starch) and certain complex silicates, combined with binders, such as polyvinylpyrrolidone, sucrose, gelatin, and acacia. Additionally, lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, may be included for tabletting purposes. Solid compositions of a similar type may be utilized as fillers in soft- and hard-filled gelatin capsules. Related materials also include lactose and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the compounds of the presently disclosed subject matter may be combined with various sweeteners, flavorings, coloring agents, emulsifying agents, and / or suspending agents, as well as diluents, such as water, ethanol, propylene glycol, glycerin, and various similar combinations thereof.

[0084] Further embodiments provided herein include liposomal formulations of the active compounds disclosed herein. Techniques for forming liposomal suspensions are known in the art. If the compound is a water-soluble salt, it can be incorporated into lipid vesicles using conventional liposome technology. In such cases, the water solubility of the active compound allows the active compound to be substantially enclosed in the hydrophilic center or core of the liposome. The lipid layer utilized can be of any conventional composition and can contain or lack cholesterol. If the active compound of interest is water-insoluble, conventional liposome formation technology can again be used to substantially enclose the salt within the hydrophobic lipid bilayer that forms the liposome structure. In either case, the liposomes produced can be reduced in size, as by standard sonication and homogenization techniques. The liposomal formulations containing the active compounds disclosed herein can be lyophilized to produce a lyophilizate, which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate the liposomal suspension.

[0085] For pharmaceutical compositions, the pharmaceutically effective amount of the active compounds described herein is determined by a healthcare professional and depends on the condition, size, and age of the patient, as well as the route of delivery. In certain non-limiting embodiments, a dosage of about 0.1 to about 200 mg / kg is therapeutically effective, where the weight ratio is the ratio of the weight of the active compound to the subject's body weight, including when a salt is used. In some embodiments, the dosage can be the amount of active compound necessary to provide a serum concentration of the active compound of up to about 1 to 5, 10, 20, 30, or 40 μM. In some embodiments, dosages of about 1 mg / kg to about 10 mg / kg, and in some embodiments, about 10 mg / kg to about 50 mg / kg, can be used for oral administration. Typically, dosages of about 0.5 mg / kg to 5 mg / kg can be used for intramuscular injection. In some embodiments, dosages can be from about 1 μmol / kg to about 50 μmol / kg, or optionally from about 22 μmol / kg to about 33 μmol / kg of the compound for intravenous or oral administration. Oral dosage forms can contain any suitable amount of active compound, including, for example, 5 mg to 50, 100, 200, or 500 mg per tablet or other solid dosage form.

[0086] Pharmaceutical compositions can utilize the active compound as a free base or as a salt. Common salts include monohydrates and hydrochloride hydrates, the latter of which may be useful for improving solubility. Exemplary pharmaceutical compositions are provided, but are intended to be non-limiting examples only. In capsule form, the compositions may contain 50 mg or 100 mg of active compound per capsule. Other ingredients include gelatin, magnesium stearate, shellac glaze, sodium lauryl sulfate, starch, quinoline yellow (E104), erythrosine (E127), patent blue V (E131), titanium dioxide (E171), triiron tetroxide (E172), and propylene glycol. The delayed-release tablet form may contain 60 mg or 120 mg of active compound, 3.6 mg or 7.2 mg of sodium, respectively, and inactive ingredients including lactose monohydrate, microcrystalline cellulose, sodium lauryl sulfate, sodium chloride, talc, anhydrous lactose, corn starch, crospovidone, magnesium stearate, and a cellulose polymer coating. It is understood that other pharmaceutical compositions may be used without departing from the inventive approach, and that the inventive approach is not intended to be limited to any particular formulation.

[0087] In some embodiments, the approaches of the present invention may take the form of a therapeutic method comprising administering to a patient in need thereof a pharmaceutically effective amount of one or more pharmaceutical compositions and a pharmaceutically acceptable carrier. For example, the approaches of the present invention may be used to eradicate a population of CSCs likely to cause metastasis, thereby preventing or reducing the likelihood of metastasis and recurrence from the original CSC population.

[0088] The following paragraphs describe the materials and methods used in connection with the data presented herein. MCF7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC). hTERT-BJ1 fibroblasts were as described in Ozsvari B, Fiorillo M, Bonuccelli G, Cappello AR, Frattaruolo L, Sotgia F, Trowbridge R, Foster R, Lisanti MP. Mitoriboscins: Mitochondrial-based therapeutics targeting cancer stem cells (CSCs), bacteria, and pathogenic yeast. Oncotarget. 2017 Jul 7;8(40):67457-67472. Cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), glutamine, and Pen / Strep.

[0089] 9-amino-doxycycline derivatives (e.g., Doxy-Myr, Doxy-Pal, Doxy-Laur, etc.) were custom synthesized. Each free fatty acid was covalently attached to 9-amino-doxycycline using conventional peptide synthesis methods. The desired reaction products were identified and purified by chromatography, and their chemical structures were verified using a combination of NMR and mass spectrometry. The IUPAC nomenclature names for these chemical compounds are as follows: Doxycycline: (4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide 9-Amino-doxycycline: (4S,5S,6R,12aS)-9-amino-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide. Note that although this is commercially available (e.g., as A14590, HCl by Frontier Scientific, Logan, Utah), 9-amino-doxycycline was essentially synthesized as previously described in Barden T. C, Buckwalter B. L, Testa R. T, Petersen P. J, Lee VJ "Glycylcyclines". 3. 9-Aminodoxycyclinecarboxamides. J. Med. Chem. 1994, 37, 3205-3211. Doxy-Myr: (4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-9-(tetradecanolamino)-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide Doxy-Laur: (4S,5S,6R,12aS)-4-(dimethylamino)-9-(dodecanolamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide Doxycycline-Pal: (4S,5S,6R,12aS)-4-(dimethylamino)-9-(hexadecanolamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide

[0090] The compound used to generate the data above was synthesized from doxycycline hydrate purchased from Alfa Aesar. The 9-amino-doxycycline derivative was synthesized following the general method for (4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-9-(tetradecanolamino)-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide. The reaction is shown below, with the individual steps being: myristic acid (R = CH3(CH2) 12 The case of Doxy-Myr using lauric acid (R = CH3(CH2)) is explained. 10 ), and Doxy-Pal was synthesized using palmitic acid (R = CH3(CH2) 14 ) Furthermore, Doxy-TPP was synthesized using R = PH3P + It was synthesized using (CH2)5.

[0091] [ka]

[0092] Step (a): To a stirred solution of doxycycline hydrate (1.0 g, 2.16 mmol) in concentrated HSO (5.5 mL) at room temperature under a nitrogen atmosphere, NaNO (0.29 g, 3.41 mmol) was added, and the mixture was stirred for 3 h. The resulting dark brown oil was poured into ice-cold diethyl ether (140 mL), and the precipitate was collected under a nitrogen atmosphere, washed with diethyl ether, and dried under vacuum to give crude 9-nitrodoxycycline.

[0093] Step (b): Crude 9-nitrodoxycycline (1.0 g, 2.04 mmol) was dissolved in methanol (30 mL) at room temperature under a nitrogen atmosphere. PtO (0.12 g) was added, and the suspension was stirred under a hydrogen atmosphere for 2 h. The catalyst was removed by filtration through a Celite pad, and the filtrate was poured into diethyl ether (240 mL) under a nitrogen atmosphere. The precipitate was collected and dried under vacuum to give crude 9-aminodoxycycline (0.89 g, 1.94 mmol).

[0094] Step (c): Crude 9-aminodoxycycline (0.70 g, 1.5 mmol), myristic acid (0.36 g, 1.5 mmol), HBTU (0.85 g, 2.25 mmol), and NMM (0.33 mL, 3.0 mmol) in a mixture of DCM (12 mL) and DMF (4 mL) were stirred at room temperature under a nitrogen atmosphere for 72 hours. The solvent was evaporated under reduced pressure. The resulting residue was triturated with acetonitrile (40 mL), and the precipitate was collected by filtration, washed with acetonitrile (10 mL), diethyl ether (20 mL), and dried under vacuum. The crude product was dissolved in DMSO and purified by preparative HPLC to give (4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-9-(tetradecanolamino)-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide (1) (0.086 g). 1H-NMR (MeOD) 0.89 (dd, 3H), 1.14-1.48 (m, 20H), 1.54 (d, 3H), 1.61-1.79 (m, 2H), 2.38-2.53 ( dd, 2H), 2.49‐2.61(m, 2H), 2.65(m, 8H), 3.68(dd, 1H), 3.94(m, 1H), 6.93(d, 1H), 8.14(d, 1H). LC‐MS 670.2[M+H]+, RT 2.78 min.

[0095] Doxy-Lauryl (4S,5S,6R,12aS)-4-(dimethylamino)-9-(dodecanolamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide (2). LC-MS 642.1 [M+H]+, RT 2.42 min.

[0096] Doxy-Pal is (4S,5S,6R,12aS)-4-(dimethylamino)-9-(hexadecanolamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide (3). LC-MS 698.2 [M+H]+, RT 3.02 min.

[0097] Doxy-TPP: [6-[[(5R,6S,7S,10aS)-9-carbamoyl-7-(dimethylamino)-1,6,8,10a,11-pentahydroxy-5-methyl-10,12-dioxo-5a,6,6a,7-tetrahydro-5H-tetracen-2-yl]amino]-6-oxo-hexyl]-triphenyl-phosphonium oxalate (4). LC-MS: 409.7 [M1 / 2]+, RT: 1.53 min.

[0098] For the 3D tumor assay, single-cell suspensions of MCF7 cells were prepared using enzymatic dissociation (1x trypsin-EDTA, Sigma-Aldrich) and manual dissociation (using a 25-gauge needle). Cells were then plated at 500 cells / cm in tumor medium (DMEM-F12 / B27 / EGF (20 ng / ml) / PenStrep) under non-adherent conditions in culture dishes coated with (2-hydroxyethyl methacrylate) (polyHEMA, Sigma). 2Cells were grown for 5 days and maintained in a humidified incubator at 37°C, atmospheric pressure, and 5% (v / v) carbon dioxide / air. After 5 days of culture, spheres larger than 50 μm were counted using an eyepiece scale, and the percentage of seeded cells that formed spheres was calculated. This is referred to as % tumor formation, normalized to vehicle-treated controls. The tumor assay was performed in triplicate and repeated three independent times.

[0099] Fluorescence imaging: Fluorescence images were taken after 72 hours of incubation of MCF7 cells treated with doxycycline or Doxy-Myr (both at 10 μM), or vehicle control. Cell cultures were imaged using the GFP channel on an EVOS Cell Imaging System (Thermo Fisher Scientific, Inc.). Because no fluorescent dye was used before imaging, any changes in signal were solely due to the autofluorescence of the doxycycline compound.

[0100] Cell viability assay: The sulforhodamine B (SRB) assay is based on the measurement of cellular protein content. After 72 hours of treatment in 96-well plates (8,000 cells / well), cells were fixed with 10% trichloroacetic acid (TCA) for 1 hour in a cold room and allowed to dry overnight at room temperature. Cells were then incubated with SRB for 15 minutes, washed twice with 1% acetic acid, and air-dried for at least 1 hour. Finally, the protein-binding dye was dissolved in 10 mM Tris pH 8.8 and read at 540 nm using a plate reader.

[0101] Cell proliferation: Briefly, MCF7 cells or hTERT-BJ1 fibroblasts were seeded into each well (10,000 cells / well) and used to evaluate the efficacy of doxycycline and Doxy-Myr using real-time cell analysis (RTCA) by measuring cell-induced electrical impedance on a plate (Acea Biosciences Inc.). This approach allows for quantification of the initiation and kinetics of the cellular response. Experiments were repeated multiple times independently, with quadruplicate samples for each condition.

[0102] Cell cycle analysis was performed on MCF7 cells treated with doxycycline, Doxy-Myr, or vehicle alone. Briefly, after trypsinization, resuspended cells were incubated with 10 ng / ml Hoechst solution at 37°C in the dark for 40 minutes. After 40 minutes, cells were washed and resuspended in PBS Ca / Mg for acquisition on an Attune NxT flow cytometer (Thermo Scientific). 10,000 events were analyzed per condition. Gated cells were manually classified into cell cycle stages.

[0103] Bacterial Growth Assays: Briefly, antibiotic activity was assessed using standard assay systems. The antibiotic activity of doxycycline analogs was determined experimentally using standard strains of Escherichia coli and Staphylococcus aureus in a 96-well plate format, using resazurin (R7017; Sigma-Aldrich, Inc.) as a probe. The minimum inhibitory concentration (MIC) of the studied compounds was determined using the broth microdilution method, a reference susceptibility test for rapidly growing aerobic or facultative microorganisms. These assays were performed in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Stock solutions of test compounds and a positive control (doxycycline, Sigma Aldrich #D1822) were prepared at 25 mM in DMSO and serially diluted (2-fold dilutions from 200 to 1.56 μM) in cation-adjusted Mueller Hinton Broth (MHB, Sigma Aldrich #90922) in a 96-well clear plate (VWR #734-2781) to a final volume of 50 μL per well. Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) cultures were grown overnight at 37°C in Mueller Hinton Agar (MHA, Sigma Aldrich #70191). One colony of each strain was then isolated at OD . 600 Grow overnight at 37 °C until the OD is approximately 0.6-0.8 and the OD is approximately 0.01. 600 The inoculum was further diluted in MHB to a concentration of 10 colony forming units (CFU) / mL, equivalent to 100 μM. Next, 50 μL of the diluted inoculum was transferred to wells of a previously prepared 96-well plate containing the test compound, a negative control (1% DMSO in MHB), and a positive control (doxycycline). The final well volume was 100 μL, the final test compound concentrations were 100-0.78 μM, and the final microbial concentration was 5 × 10 5CFU / mL. Subsequently, 10 μL of one negative control well was plated onto a Petri dish containing MHA to check the CFU and purity of the culture. After incubating the plate at 37°C for 24 hours, 20 μL of resazurin solution (0.2 mg / mL) was added to the well and then incubated at 37°C for 1 hour and 30 minutes. OD 570 and O.D. 600 OD measured with a microplate reader (BMG FLUOstar Omega) 570 and O.D. 600 The MIC represents the lowest concentration of a compound that inhibited bacterial growth (OD 570 / OD 600 (The ratio is lower than the average ratio determined for the negative control wells.) MIC values ​​were determined in three independent experiments.

[0104] Tumor growth, metastasis, and embryotoxicity assays: These xenograft assays were performed without major modifications. a) Chicken embryo preparation. Fertilized White Leghorn eggs were incubated at 37.5°C and 50% relative humidity for 9 days. At this time (E9), the chorioallantoic membrane (CAM) was removed by puncturing a small hole through the eggshell into the air sac, and a 1 cm thick layer of the eggshell above the CAM was placed. 2 I opened the window. b) Tumor cell expansion and transplantation. MDA-MB-231 tumor cell line was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. On day E9, cells were detached with trypsin, washed with complete medium, and suspended in transplantation medium. 1 × 10 6 An inoculum of 10 cells was added to the CAM of each egg (E9), after which the eggs were randomly divided into groups. c) Tumor growth assay. On day 18 (E18), the top of the CAM was removed from each egg, washed with PBS, and then directly transferred to paraformaldehyde (fixed for 48 hours) and weighed. For tumor growth assays, at least 10 tumor samples per group were collected and analyzed (n≧10). d) Metastasis assay. On day E18, 1 cm of the lower CAM was implanted in 8 samples per group (n=8). 2 Portions of the CAM were harvested and the number of metastatic cells assessed. Genomic DNA was extracted from the CAM (using a commercial kit) and analyzed by qPCR using primers specific for the human Alu sequence. Calculation of the Cq for each sample, the mean Cq, and the relative amount of metastasis in each group were directly managed by Bio-Rad® CFX Maestro software. All data were subjected to one-way ANOVA with post-hoc testing. e) Embryo Tolerance Assay: Before each administration, the tolerability of the treatment was assessed by scoring the number of dead embryos.

[0105] Statistical analysis: Statistical significance was determined using Student's t-test, with values ​​less than 0.05 considered significant. Data are presented as mean ± SEM unless otherwise stated.

[0106] The terminology used in the above description of embodiments of the inventive approach is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The inventive approach encompasses numerous alternatives, modifications, and equivalents, as will become apparent from a discussion of the following Detailed Description.

[0107] The terms "first," "second," "third," "a)," "b)," "c)," etc. may be used herein to describe various elements of the inventive approach, and it will be understood that the claims are not limited by these terms. These terms are used only to distinguish one element of the inventive approach from another. Thus, a first element described below could be referred to as an element aspect, or similarly, as a third element, without departing from the teachings of the inventive approach. Thus, the terms "first," "second," "third," "a)," "b)," "c)," etc. are not intended to impose any order or other hierarchical relationship to the associated elements, but are used merely for identification purposes. The order of operations (or steps) is not limited to the order presented in the claims.

[0108] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, it will be understood that terms as defined in commonly used dictionaries shall be interpreted as having a meaning consistent with the meaning of said terms in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into this application. In the event of a conflict in terminology, the present specification shall control.

[0109] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted as alternatives ("or").

[0110] Unless the context clearly dictates otherwise, it is specifically intended that the various features of the inventive approach described herein can be used in any combination. Furthermore, the inventive approach contemplates that, in some embodiments, any feature or combination of features described with respect to the illustrative embodiments can be excluded or omitted.

[0111] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) should be interpreted to include the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. Thus, as used herein, the term "consisting essentially of" should not be interpreted as equivalent to "comprising."

[0112] The term "about," as used herein in reference to a measurable value, such as an amount or concentration, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even no more than ±0.1% of the specified amount. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​within that range.

[0113] Thus, while particular embodiments of the inventive approach have been described, it should be understood that the appended claims are not limited to the specific details set forth in the foregoing description, as many obvious variations of these embodiments are possible without departing from the spirit or scope of the invention as hereinafter claimed.

Claims

1. of the following general formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein R is a straight chain saturated alkyl having 11 to 16 carbons.

2. 2. The compound of claim 1, wherein R is a straight chain saturated alkyl having 14 carbons.

3. The compound is: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

4. The compound is: 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

5. The compound is: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

6. 10. The compound of claim 1, which is a pharmaceutically acceptable salt, said salt being one of a monohydrate and a hydrochloride hydrate.

7. A pharmaceutical composition for preventing metastasis of cancer, said composition comprising a compound of the general formula: 【Transformation 5】 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein R is a straight chain saturated alkyl having 11 to 16 carbons.

8. 8. The pharmaceutical composition of claim 7, wherein R is a straight chain saturated alkyl having 14 carbons.

9. The compound is: 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

10. The compound is: 【Transformation 7】 or a pharmaceutically acceptable salt thereof.

11. The compound is: 【Transformation 8】 or a pharmaceutically acceptable salt thereof.

12. 8. The pharmaceutical composition of claim 7, comprising a pharmaceutically acceptable salt, said salt being one of a monohydrate and a hydrochloride hydrate.

13. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable carrier comprises at least one of a sugar, a starch, a cellulose, an excipient, an oil, a glycol, a polyol, an ester, an agar, and a buffer.

14. 14. The pharmaceutical composition of any one of claims 7 to 13 for use in one of preventing cancer metastasis, reducing inflammation, reducing fibrosis, and reducing viral replication.

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